The mammalian organism contains millions of distinct T cells, each with their own unique specificity. T cells are essential components of the adaptive immune response, being critical to the defense of our bodies against foreign pathogens and tumors that create cellular alterations. The T cell receptor (TCR), a multimeric transmembrane complex, is highly adaptive, capable of distinguishing foreign peptides bound to self- major histocompatibility complex molecules (pMHC) displayed on the surface of altered cells. External flags in the form of these presented peptides displayed on antigen presenting cells are sensed and transmitted into the T cell during its process of immune surveillance. Once recognized by the T cell, immune activation is initiated to destroy the non-self invader. How recognition of ths peptide by a weakly interacting specific receptor on the T cell surface evokes signaling remains an undefined, longstanding, biological question. Our preliminary studies identified via focused experimentation the importance of physical forces in activating T cells, providing a mechanical mechanism to answer the long standing biological question of how minute differences at the TCR pMHC interface lead to triggering. Our objective is to test the hypothesis that the TCR acts as an anisotropic mechanosensor. The concept that mechanical force plays a role in TCR activation will be explored across physiologically relevant model cell-based systems as well as be adapted to classical single molecule assay design. We combine single molecule methods, structure function mutational analysis, and recombinant protein expression specifically targeted to the T cell triggering event. First, we determine the physical and chemical requirements for TCR triggering on cells. Second, we develop a single molecule assay for probing the strength of the TCR-pMHC bond across a range of loads with peptides exhibiting varying potencies. Third, we determine the role of the stalk domain as a force transducer for T cell triggering in live T cells. Outcome of this work will provide a clearer understanding for the temporal, chemical, spatial and physical inputs to TCR mechanosensing. Our project includes training of personnel at levels ranging from undergraduates to faculty with crucial interdisciplinary overlap between labs founded in single molecule measurement and immunology disciplines. PUBLIC HEALTH RELEVANCE: A deeper understanding of the adaptive immune response is critical to understanding how our bodies recognize and destroy foreign pathogens and tumors. Our work combines single molecule methods, structure function mutational analysis, and recombinant protein expression for investigating immune activation.